Splenic Th17 Research Articles

Analysis of the role of palmitoleic acid in acute anterior uveitis

PurposeTo study the role of palmitoleic acid (PA) in the pathogenesis of acute anterior uveitis (AAU). MethodsPA levels in feces from AAU patients were measured by gas chromatography coupled with a mass spectrometer (GC-MS) and compared with samples obtained from healthy individuals. Enzyme linked immunosorbent assay (ELISA) and flow cytometry (FCM) were used to assess the effect of PA on dendritic cells (DCs) and CD4+T cells obtained from mice, AAU patients and healthy individuals. C57BL/6 mice were fed with PA or vehicle and experimental autoimmune uveitis (EAU) was induced with a human retinal IRBP651-670 peptide. Disease severity of EAU was evaluated by clinical manifestation and histology. Differentiation of splenic Type 1 helper T cells (Th1) and Th17 cells was evaluated by FCM. Tandem mass tag (TMT)-based proteomics analysis was used to identify differentially expressed proteins following incubation of DCs with PA. ResultsThe fecal concentration of PA was increased in AAU patients as compared with healthy individuals. In vitro, PA promoted apoptosis of DCs and inhibited the secretion of TNF-α from mouse bone-marrow-derived dendritic cells (BMDCs) as well as in DCs from AAU patients and healthy individuals. It only decreased DCs surface marker expression and IL-12p70 secretion in BMDCs and healthy individuals DCs but not in AAU patient DCs. PA-treated BMDCs inhibited Th cell differentiation from mouse naïve CD4+T cells and IL-17 and IFN-γ secretion in co-culture supernatants. PA also inhibited the differentiation of Th cells and secretion of IFN-γ and IL-17 in CD4+T cells from mice, AAU patients and healthy individuals. In vivo, PA-treated EAU mice showed milder clinical and histopathological intraocular manifestations as compared with the control group. PA feeding inhibited differentiation of splenic Th17 cells, whereas Th1 cells were not affected. Up to 30 upregulated and 77 downregulated proteins were identified when comparing PA-treated DCs with controls. ConclusionAn increased expression of fecal PA was observed in AAU patients. PA was shown to have immunoregulatory effects on DCs and CD4+T cells and attenuated disease severity in EAU mice.

Oncolytic Newcastle disease virus delivered by Mesenchymal stem cells-engineered system enhances the therapeutic effects altering tumor microenvironment

BackgroundHuman papillomavirus (HPV)-associated malignancy remain a main cause of cancer in men and women. Cancer immunotherapy has represented great potential as a new promising cancer therapeutic approach. Here, we report Mesenchymal stem cells (MSCs) as a carrier for the delivery of oncolytic Newcastle disease virus (NDV) for the treatment of HPV-associated tumor.MethodsFor this purpose, MSCs obtained from the bone marrow of C57BL mice, then cultured and characterized subsequently by the flow cytometry analysis for the presence of cell surface markers. In this study, we sought out to determine the impacts of MSCs loaded with oncolytic NDV on splenic T cell and cytokine immune responses, caspase-3 and -9 expression, and myeloid and myeloid-derived suppressor cells (MDSCs) by histological and immunohistochemical studies in the tumor microenvironment (TME).ResultsOur findings proved that MSCs possess both migratory capacity and tumor tropism toward transplanted tumor tissue after peritumoral administration. Tumor therapy experiments indicated that oncolytic NDV delivered by MSCs-engineered system significantly reduces tumor growth, which is associated with the enhancement of E7-specific lymphocyte proliferation, CD8+ T cell cytolysis responses, and splenic IFN-γ, IL-4 and IL-12 responses compared with control groups. Moreover, the treatment upregulated the concentration of apoptotic proteins (caspase 9) and increased infiltration of tumor microenvironment with CD11b + myeloid and Gr1 + MDSCs cells.ConclusionsOur data suggest MSCs carrying oncolytic NDV as a potentially effective strategy for cancer immunotherapy through inducing splenic Th1 immune responses and apoptosis in the tumor microenvironment.

RNA-Binding Protein HuR Promotes Th17 Cell Differentiation and Can Be Targeted to Reduce Autoimmune Neuroinflammation.

Dysregulated Th17 cell differentiation is associated with autoimmune diseases such as multiple sclerosis, which has no curative treatment. Understanding the molecular mechanisms of regulating Th17 cell differentiation will help find a novel therapeutic target for treating Th17 cell-mediated diseases. In this study, we investigated the cell-intrinsic processes by which RNA-binding protein HuR orchestrates Th17 cell fate decisions by posttranscriptionally regulating transcription factors Irf4 and Runx1 and receptor Il12rb1 expression, in turn promoting Th17 cell and Th1-like Th17 cell differentiation in C57BL/6J mice. Knockout of HuR altered the transcriptome of Th17 cells characterized by reducing the levels of RORγt, IRF4, RUNX1, and T-bet, thereby reducing the number of pathogenic IL-17+IFN-γ+CD4+ T cells in the spleen during experimental autoimmune encephalomyelitis. In keeping with the fact that HuR increased the abundance of adhesion molecule VLA-4 on Th17 cells, knockout of HuR impaired splenic Th17 cell migration to the CNS and abolished the disease. Accordingly, targeting HuR by its inhibitor DHTS inhibited splenic Th17 cell differentiation and reduced experimental autoimmune encephalomyelitis severity. In sum, we uncovered the molecular mechanism of HuR regulating Th17 cell functions, underscoring the therapeutic value of HuR for treatment of autoimmune neuroinflammation.

Aminooxy-Acetic Acid Inhibits Experimental Autoimmune Uveitis by Modulating the Balance between Effector and Regulatory Lymphocyte Subsets.

A small molecular compound, aminooxy-acetic acid (AOA), has been shown to modulate experimental autoimmune encephalomyelitis (EAE). The current study was designed to investigate whether AOA has a similar effect on the development of experimental autoimmune uveitis (EAU) and to further explore underlying mechanisms of this drug. EAU was induced in C57BL/6J mice by immunization with interphotoreceptor retinoid-binding protein peptide 651-670 (IRBP 651-670). AOA (500μg or 750μg) or vehicle was administered by intraperitoneal injection from day 10 to 14 after EAU induction. The severity was assessed by clinical and histological scores. The integrity of the blood retinal barrier was detected with Evans Blue. Frequencies of splenic Th1, Th17 and Foxp3+ Treg cells were examined by flow cytometry. The production of cytokines was tested by ELISA. The mRNA expression of IL-17, IFN-γ and IL-10 was detected by RT-PCR. The expression of p-Stat1 and NF-κB was detected by Western Blotting. AOA was found to markedly inhibit the severity of EAU, as determined by clinical and histopathological examinations. AOA can relieve the leakage of blood retinal barrier (BRB). Functional studies found a decreased frequency of Th1 and Th17 cells and an increased frequency of Treg cells in EAU mice as compared with controls. Further studies showed that AOA not only downregulated the production of the proinflammatory cytokines including IFN-γ and IL-17 but also upregulated the expression of an anti-inflammatory cytokine such as IL-10, which might be caused by inhibiting the expressions of p-Stat1 and NF-κB. This study shows that AOA inhibits the severity and development of EAU by modulating the balance between regulatory and pathogenic lymphocyte subsets.

Standardized Xylocarpus moluccensis fruit fraction mitigates collagen-induced arthritis in mice by regulating immune response

This study was undertaken to evaluate the effect of Xylocarpus moluccensis fruit fraction (F018) on the pathogenesis of collagen-induced arthritis in mice. Arthritis was induced by intradermal injection of collagen (2mg/ml) with complete Freund's adjuvant in DBA/1J mice. F018 was administered orally at 1, 3 and 10mg/kg for 20days. Disease progression and mechanism were assessed by micro-CT analysis, RT-PCR, flow cytometry assay, myeloperoxidase (MPO) and MTT assay. F018 at 3 and 10mg/kg significantly reduced paw thickness, clinical score, mononuclear cell infiltration and collagen layer depletion in the knee section of collagen-induced arthritis (CIA) mice when compared with collagen-induced arthritis mice alone. Furthermore, F018 treatment in collagen-induced arthritis mice significantly recovered bone volume and trabecular number and decreased the trabecular space by modulating RANKL and OPG mRNA expression in the synovial tissue. F018 treatment in collagen-induced arthritis mice significantly attenuated spleen index, lymphocyte proliferation and paw myeloperoxidase (MPO) activity, pro-inflammatory cytokine TNFα, IL1β, and IL6 mRNA expression and enhanced IL10 mRNA expression in paw tissue. Furthermore, F018 treatment in collagen-induced arthritis mice significantly reduced splenic dendritic cell maturation and Th17 cells. In culture, F018 significantly decreased collagen-induced arthritis-FLS proliferation and promoted apoptosis. F018 may serve as a potential curative agent for arthritis.

Effect of berberine on spleen transcriptome and gut microbiota composition in experimental autoimmune uveitis

BackgroundBerberine (BBR) was reported to have immunoregulatory and anti-inflammatory properties. In this study, we investigated whether BBR could exert its effects on the development of experimental autoimmune uveitis (EAU), and if so, what was the underlying mechanism? MethodsEAU was induced in B10R.III mice by immunization with IRBP 161–180, followed by 100 mg/kg/d BBR intragastric administration. Disease severity was assessed by evaluation of clinical and histopathological scores. Blood-retinal barrier (BRB) breakdown was tested by Evans blue. Effector and regulatory T (Treg) cell balance was evaluated by quantitative real-time PCR and flow cytometry. Spleen transcriptome was characterized by RNA sequencing (RNA-seq). Gut microbiota composition was investigated by 16S rRNA analysis. ResultsBBR treatment significantly blocked EAU as shown by the decrease of the clinical and histological scores, as well as the inhibition of BRB breakdown. The frequency of splenic Th1 and Th17 cells was decreased, whereas Treg cells were increased in the BBR-treated group. RNA-seq of the spleen revealed 476 differentially expressed genes (DEGs) between the EAU and EAU-BBR group. GO functional classification, as well as KEGG analysis demonstrated that BBR treatment markedly influences genes belonging to chromatin remodeling and immune-related pathways. Intervention with BBR modified the gut microbiome in EAU mice, increasing the number of bacteria with immunomodulatory capacity. Depletion of gut microbiota affected the efficacy of BBR on EAU. Moreover, the altered bacterial strains showed a significant correlation with the expression of histones. ConclusionsBBR inhibited IRBP induced EAU, which was associated with a significant change in the spleen transcriptome and intestinal microbial composition.

The environmental hormone nonylphenol interferes with the therapeutic effects of G protein-coupled estrogen receptor specific agonist G-1 on murine allergic rhinitis

The G protein-coupled estrogen receptor (GPER) specific agonist G-1 has therapeutic effects in patients with allergic diseases, but any role for G-1 as a therapy for inflammation associated with allergic rhinitis (AR) remains unclear. The structure of the environmental hormone nonylphenol (NP) is very similar to that of estrogen; it binds to the estrogen receptor to produce estrogen-like effects and thus may also bind to the membrane GPER. We explored whether NP administration would reduce the effects of G-1 on AR, the interactions between the two materials, and their mechanisms of action using a murine model of AR. Mice were randomly assigned into control, AR, G-1, and G-1 + NP groups (n = 10/group). AR nasal symptoms were scored. Eosinophils in nasal mucosa were counted after staining with hematoxylin and eosin. Serum ovalbumin (OVA)-specific IgE was determined by ELISA. The proportions of splenic Th1, Th2, and Treg cells were determined by flow cytometry. The expression of transcription factors unique to Th1, Th2, Treg cells and cytokine levels in nasal mucosa were evaluated by real-time PCR and cytometric bead arrays. AR nasal symptoms, including sneezing, nasal scratching, eosinophil infiltration of nasal mucosa, and serum IgE, were reduced in G-1 group. After injection, Th2 cells proportions, Th2-immune response-related cytokines (IL-4, IL-5, and IL-13), and a Th2 cell-specific transcription factor (GATA-3) were significantly decreased in G-1 group. Treg immune response was enhanced (as reflected by Treg cell, IL-10, and Foxp3 levels). The levels of all of these were significantly increased after adding NP, and the Treg immune response was significantly decreased. These results indicate that G-1 attenuated the nasal symptoms, serum OVA-specific IgE, and Th2 cell immune response, whereas it enhanced Treg immune response, in mice with AR. Adding NP weakened these therapeutic effects.

Deletion of CD137 Ligand Exacerbates Renal and Cutaneous but Alleviates Cerebral Manifestations in Lupus.

The CD137—CD137 ligand (CD137L) costimulatory system is a critical immune checkpoint with pathophysiological implications in autoimmunity. In this study, we investigated the role of CD137L-mediated costimulation on renal, cutaneous and cerebral manifestations in lupus and the underlying immunological mechanism. Lupus-prone C57BL/6lpr−/− (B6.lpr) mice were crossed to C57BL/6.CD137L−/− mice to obtain CD137L-deficient B6.lpr [double knock out (DKO)] mice. We investigated the extent of survival, glomerulonephritis, skin lesions, cerebral demyelination, immune deviation and long-term synaptic plasticity among the two mouse groups. Cytokine levels, frequency of splenic leukocyte subsets and phenotypes were compared between DKO, B6.lpr and B6.WT mice. A 22 month observation of 226 DKO and 137 B6.lpr mice demonstrated significantly more frequent proliferative glomerulonephritis, larger skin lesions and shorter survival in DKO than in B6.lpr mice. Conversely, microglial activation and cerebral demyelination were less pronounced while long-term synaptic plasticity, was superior in DKO mice. Splenic Th17 cells were significantly higher in DKO than in B6.lpr and B6.WT mice while Th1 and Th2 cell frequencies were comparable between DKO and B6.lpr mice. IL-10 and IL-17 expression by T cells was not affected but there were fewer IL-10-producing myeloid (CD11b+) cells, and also lower serum IL-10 levels in DKO than in B6.lpr mice. The absence of CD137L causes an immune deviation toward Th17, fewer IL-10-producing CD11b+ cells and reduced serum IL-10 levels which potentially explain the more severe lupus in DKO mice while leading to reduced microglia activation, lesser cerebral damage and less severe neurological deficits.

Renal TH17 cells are Elevated in Humanized Sickle Cell Mice

Sickle cell nephropathy (SCN) is a major complication of sickle cell disease (SCD). However, it is not fully understood what role immune cells play in SCN. Recent studies have implicated IL‐17A+CD4+(TH17) cells in mediating kidney disease outside of SCD. Furthermore, levels of circulating TH17 cells are elevated in SCD patients. We have previously reported that humanized sickle cell (HbSS) mice show progressive age‐dependent nephropathy with proteinuria, glomerulosclerosis, loss of GFR, and elevated CD3+cells compared to control (HbAA) mice. However, a role for renal TH17 cells in SCN has not yet been defined. We hypothesized that HbSS mice have elevated renal TH17 cells compared to HbAA mice. Thus, we phenotyped renal and splenic lymphocytes from 16–24 week‐old male HbAA and HbSS mice by flow cytometry. HbSS mice had increased renal TH17 cells compared to HbAA mice (18.3 ± 2.0% vs. 0.2 ± 0.1%, respectively; p<0.001) but no difference in renal CD4+cells. No differences were observed in splenic TH17 cells between HbSS and HbAA mice, which correlated with similar frequencies of splenic CD4+cells. Interestingly, HbSS mice had a significant increase in renal CXCR3+CD4+(TH1) cells compared to HbAA mice (48.8 ± 2.3% vs. 10.9 ± 3.3%, respectively; p<0.001) but no difference in splenic TH1 cells. Next, we measured changes in circulating proinflammatory mediators by Luminex multiplex technology. HbSS mice had elevated levels of soluble intercellular adhesion molecule 1 (sICAM‐1: 2937 ± 240 ng/mL vs. 2396 ± 57 ng/mL, respectively; p=0.04 ) and plasminogen activator inhibitor 1 (PAI‐1: 546.9 ± 120 ng/mL vs. 69.6 ± 4.5 ng/mL; p=0.001 ) compared to HbAA mice. We conducted a preliminary flow cytometric analysis of activated renal and splenic CD4+cells from aged (8–13 month‐old) mice and observed decreased splenic CD4+cells in aged HbSS mice compared to aged HbAA mice (5.1% vs. 32.5%, respectively; n = 2) but no differences in renal CD4+cells. Activated CD44hiCD4+cells from aged HbSS mice were increased in both the spleen (68.8% vs. 30.8%; n = 2) and the kidney (22.2% vs. 14.0%, respectively; n = 2) compared to aged HbAA mice. We propose that these findings and initial observations suggest an important role for renal T cells, especially TH17 and TH1 cells, in promoting and exacerbating kidney injury and dysfunction in SCD.Support or Funding InformationSupport provided by NIH T32 DK116672 to PAM, F30 DK107194 to BMF, NIH P01 HL136267 to CDM, JSP, DMP, NIH T32 AR069516 to RJM, NIH U01 HL117684 to JSP, DMP, and ASN Ben J. Lipps Research Fellowship to MK.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Adoptive transfer of regulatory T cells stimulated by Allogeneic Hepatic Stellate Cells mitigates liver injury in mice with concanavalin A-induced autoimmune hepatitis

Regulatory T cell (Treg)-based therapy can effectively control autoimmune hepatitis (AIH). Hepatic stellate cells (HSC) can selectively stimulate allogeneic Treg proliferation following liver transplantation. This study tested the therapeutic effect and potential mechanisms underlying the action of HSC-stimulated Tregs on AIH in a mouse model of Concanavalin A (ConA)-induced AIH. HSC were isolated from BALB/c mice and characterized. Splenic CD4+CD25+ Tregs were isolated from C57BL/6 mice by immunomagnetic beads. The cells were co-cultured with primary (HSC-0), the second generation of HSC (HSC-2) for 72 h. The proliferation of Tregs was determined by flow cytometry. Similarly, the Tregs were co-cultured with HSC in transwell plates to determine the potential cell-cell contact dependent. The CD4+CD25− effector T cells (CFSE-Teffs) were co-cultured with Teff or Tregs in the presence or absence of HSC to determine the suppressive capacity of Tregs. The effects of Tregs or HSC-stimulated Tregs on AIH severity and the frequency of splenic Tregs and Th17 cells were examined in mice. Co-culture with HSC-2 significantly promoted Treg proliferation in a dose- and cell-cell contact-dependent manner, and allogeneic HSC enhanced the suppressive activity of Tregs to inhibit the proliferation of Teff in vitro. Adoptive transfer of Tregs, particularly of HSC-stimulated Tregs, significantly reduced liver injury, inflammation and Ishak modified histology activity index in AIH mice, which were associated with improving the balance of Treg and Th17 cell responses. Our data indicated that mature HSC stimulated allogeneic Treg proliferation in a dose and cell-cell contact-dependent manner, and HSC enhanced the suppressive activity of Tregs to inhibit the proliferation of Teff. Adoptive transfer of HSC-stimulated Tregs significantly reduced liver injury in AIH mice by modulating the balance of Treg and Th17 cell responses.

Amlexanox attenuates experimental autoimmune encephalomyelitis by inhibiting dendritic cell maturation and reprogramming effector and regulatory T cell responses

BackgroundAmlexanox (ALX), a TBK1 inhibitor, can modulate immune responses and has anti-inflammatory properties. To investigate its role in regulating the progression of experimental autoimmune encephalomyelitis (EAE), we studied the effect of ALX on the maturation of dendritic cells (DCs) and the responses of effector and regulatory T cells (Tregs).MethodsIn vitro, bone marrow-derived DCs (BMDCs) were cultured and treated with ALX. Their proliferation, maturation, and their stimulatory function to induce T cells responses were detected. In vivo, the development of EAE from different groups was recorded. At the peak stage of disease, HE, LFB, and electronic microscope (EM) were used to evaluate inflammation and demyelination. Maturation of splenic DC and Th1/Th17/Treg response in the CNS and peripheral were also detected. To further explore the mechanism underlying the action of ALX in DC maturation, the activation of TBK1, IRF3, and AKT was analyzed.ResultsOur data indicated that ALX significantly inhibited the proliferation and maturation of BMDCs, characterized by the reduced MHCII, a co-stimulatory molecule, IL12, and IL-23 expression, along with morphological alterations. Co-culture of ALX-treated BMDCs inhibited allogeneic T cell proliferation and MOG-specific T cell response. In EAE mice, ALX significantly attenuated the EAE development by decreasing inflammatory infiltration and demyelination in the spinal cords, accompanied by reduced frequency of splenic pathogenic Th1 and Th17 cells and increased Tregs. Moreover, ALX treatment decreased Th1 and Th17 cytokines, but increased Treg cytokines in the CNS and spleen. Notably, ALX treatment reduced the frequency and expression of CD80 and CD86 on splenic DCs and lowered IL-12 and IL-23 secretion, further supporting an impaired maturation of splenic DCs. In addition, ALX potently reduced the phosphorylation of IRF3 and AKT in BMDC and splenic DCs, both of which are substrates of TBK1 and associated with DC maturation.ConclusionsALX, a TBK1 inhibitor, mitigated EAE development by inhibiting DC maturation and subsequent pathogenic Th1 and Th17 responses while increasing Treg responses through attenuating the TBK1/AKT and TBK1/IRF3 signaling.

Study of the adoptive immunotherapy on rheumatoid arthritis with Thymus-derived invariant natural killer T cells

BackgroundThe therapeutic effect of adoptive infusion of specific thymus-derived invariant natural killer T (iNKT) cells in a mouse model of rheumatoid arthritis (RA) was observed, and the mechanism of cellular immunotherapy was preliminarily explored. MethodsThymus-derived iNKT cells were infused to RA model mice, with α-GalCer as a positive control. Then, ankle swelling was examined, as well as inflammatory cell infiltration to the joint tissue (hematoxylin-eosin [H&E] staining). Flow cytometry (FCM) was used to assess iNKT cell and helper T lymphocyte (Th) subsets. Serum cytokine levels were determined with cytometric bead array (CBA), with protein expression levels of related transcription factors assessed by Western blot. ResultsThe joint swelling in RA model animals were significantly improved in the cell therapy and α-GalCer positive control groups (P < 0.05). In addition, iNKT frequencies in peripheral blood, the thymus and spleen were increased significantly (P < 0.05). Meanwhile, iNKT1 subset frequencies in the thymus and spleen were decreased, as well as splenic Th1 and Th17 cell subset rates, and serum TNF-α, IFN-γ and IL-6 levels. The rates of iNKT2 and Th2 subsets as well as IL-4 and IL-10 levels were increased (P < 0.05). Thymus GATA-3 and splenic PLZF protein levels were increased (P < 0.05). ConclusionsAdoptive infusion of thymus-derived iNKT cells exerts therapeutic effects in RA mice by increasing iNKT frequency, altering the proportions of iNKT cell subsets, correcting Th cell subset imbalance and reducing the amounts of inflammatory cytokines.

Abstract 131: Microbiota-Derived Metabolite Propionate Protects From Hypertensive Cardiovascular Damage

Objective: Inflammation drives cardiovascular disease, anti-inflammatory approaches may be beneficial. Short-chain fatty acids (SCFA) are bacterial metabolites with anti-inflammatory properties affecting host immune homeostasis including regulatory T cells (Treg). We investigated effects of the SCFA propionate (administered in drinking water, NaCl as control) in two mouse models, namely hypertensive heart disease (wild-type NMRI (WT), angiotensin (Ang)II infusion 1.44 mg/kg/d s.c. for 14 days) and atherosclerosis (Apolipoprotein E knockout (ApoE), AngII infusion 0.72 mg/kg/d s.c. for 28 days), respectively. Results: Propionate attenuated cardiac hypertrophy and fibrosis in both models significantly. Susceptibility to cardiac ventricular arrhythmias was significantly reduced in propionate-treated WT mice. Aortic atherosclerotic lesion area was significantly reduced in propionate-treated ApoE (27.6±8 vs. 7.9±2.4%). Treatment reduced splenic effector memory (CD4+ CD44+ CD62L-) T cell frequencies (WT: 30.5±4.6 vs. 19.1±1.6; ApoE: 41.1±3.1 vs. 32.7±1.4%) and splenic Th17 cells (WT: 1.0±0.2 vs. 0.6±0.1; ApoE: 1.3±0.1 vs. 0.9±0.1%) in both models, indicating beneficial effects on systemic inflammation. Similarly, propionate reduced cardiac immune cell infiltration (CD4+, CD8+, F4/80+) in WT mice. Propionate improved vascular dysfunction and moderately reduced blood pressure in both models. Organ-protective actions of propionate (cardiac inflammation and fibrosis) were abrogated in Treg-depleted (antiCD25-treated) AngII-infused WT mice, suggesting a central role for Treg. To verify our findings in a human cohort, we re-analyzed clinical and metagenomic data from a recent randomized controlled trial investigating the effect of a 90-day synbiotic intervention in 84 subjects with metabolic syndrome including healthy controls. Interestingly, in synbiotic-treated subjects an increased capacity for SFCA production was significantly correlated to blood pressure reduction. Conclusion: Data underscore the importance of SCFA for cardiovascular health and suggest that lifestyle modifications leading to augmented SCFA production could be a beneficial non-pharmacological add-on strategy for cardiovascular disease.

Plasma Cell Alloantigen 1 and IL-10 Secretion Define Two Distinct Peritoneal B1a B Cell Subsets With Opposite Functions, PC1high Cells Being Protective and PC1low Cells Harmful for the Growing Fetus.

B cells possess various immuno regulatory functions. However, research about their participation in tolerance induction toward the fetus is just emerging. Accumulating evidence supports the idea that B cells can play seemingly conflicting roles during pregnancy, either protecting or harming the fetus. Previous findings indicated the presence of two different peritoneal B cell subsets, defined by the expression of the plasma cell alloantigen 1 (PC1) and with distinct immune modulatory functions. Here, we aimed to study the participation of these two B cell subsets, on pregnancy outcome in a murine model of disturbed fetal tolerance. The frequencies and cell numbers of peritoneal and splenic CD19+IL-10+ and CD19+CD5+IL-10+PC1+ cells were assessed in virgin as well as normal pregnant (NP) and abortion-prone (AP) females during the course of gestation. Peritoneal PC1low or PC1high B1a B cells were sorted, analyzed for their ability to secrete IL-10 and adoptively transferred into NP or AP females. On gestation day (gd) 12, the abortion rate as well as the frequencies and cell numbers of regulatory T cells, TH1 and TH17 cells were determined in spleens and decidua. In addition, mRNA expression of IL-10, TGF-β, IFN-γ, and TNF-α was analyzed in decidual tissue. Peritoneal CD19+IL-10+ and CD19+CD5+IL-10+PC1+ frequencies fluctuated during the progression of normal pregnancies while no significant changes were observed in spleen. AP females showed significantly reduced frequencies of both B cell populations and exhibited an altered peritoneal PC1high/PC1low ratio at gd10. Adoptive transfers of PC1low B1a B cells into NP females increased the abortion rate in association with a reduced splenic regulatory T/TH17 ratio. By contrast, the transfer of PC1high B1a B cells into AP females significantly diminished the fetal rejection rate and significantly reduced the numbers of splenic TH17 cells. Our results suggest that the peritoneum harbors two distinct B1a B cell subsets that can be distinguished by their PC1 expression. Whereas PC1high B1a B cells seem to support fetal survival, PC1low cells B1a B cells may compromise fetal well-being.

Investigating the role of CX3CR1 in a GITRL- dependent signal 4 checkpoint during LCMV clone 13 infection

Abstract T cell-APC interactions early during chronic viral infection are critical for determining viral set point and disease outcome. Our recent work (Chang et al. Immunity. 2017) revealed a division of labour whereby monocyte-derived APCs (infAPCs), with low MHC II and CD80/86 expression compared to classical DCs (cDCs), preferentially up-regulate TNF receptor superfamily ligands, GITRL, 4-1BBL, OX40L and CD70, in response to type I interferon (signal 3) to provide a post-priming checkpoint (signal 4) for CD4 T cell responses. While cDCs contribute to signal 1 &amp; 2 during priming in response to LCMV clone 13, GITRL on infAPCs binding to GITR on CD4 T cells results in increased surface expression of pro-survival receptors, including CD25, CD127 and OX40 to sustain Th1 helper cell accumulation, help for virus-specific CD8 T cell responses and viral control. Interestingly, CX3CR1, the receptor for fractalkine, was also upregulated on CD4 T cells by GITR signalling during clone 13 infection. CX3CR1 was recently shown to contribute to tumor-infiltration of CD4-helped CD8 CTLs (Ahrends et al. Immunity. 2017). In the chronic LCMV model, we observed that CX3CR1high virus-specific CD4 T cells in the spleen exhibit a markedly increased effector profile when compared to their CX3CR1int-low counterparts, with both higher frequency and per cell expression of Tbet and IFNg production. To investigate how CX3CR1 on CD4 T cells contributes to CD4 T cell help for the CD8 T cell response, we have crossed CX3CR1 deficient mice with TCR transgenic SMARTA mice, expressing an MHC II-restricted TCR specific for LCMV gp61–80. Overall, our data so far indicate that upregulation of CX3CR1 marks the most activated splenic CD4 Th1 effectors during chronic LCMV infection.

Effect of \u03b3-secretase inhibitor on Th17 cell differentiation and function of mouse psoriasis-like skin inflammation

BackgroundTh17 cells and its effective cytokine IL-17A play an important role in the pathogenesis of abnormal immune responses in psoriasis. Notch1 signaling has been implicated in Th17 cell differentiation and function. In this study, our aim was to evaluate the possible inhibitory effect of Notch1 signaling inhibitor, γ-secretase inhibitor DAPT, on psoriatic Th17 cell differentiation and function in a mouse model of psoriasis-like skin inflammation.MethodsMouse psoriasis-like skin inflammation model was established by topical 5% imiquimod (IMQ) application, and experimental mice were divided into control group, IMQ-treated group and IM + DAPT-treated group. DAPT and the equivalent amount of Dimethyl sulfoxide was intraperitoneally injected in IMQ + DAPT-treated group and the other two experimental groups respectively. Skin tissues of the three experimental groups were acquired and stained with haematoxylin and eosin (HE). Splenic single-cells and serum were collected to detect the percentage of Th17 cells, the mRNA expression levels of Notch1 and its target gene Hes-1, Th17-specific transcription factor RORγt and its effective cytokines IL-17A, as well as IL-17A serum concentration. In addition, splenic CD4+ T cells from IMQ-treated mice were isolated and treated by DAPT to further measure the inhibitory effect of DAPT on the Th17 cell differentiation and IL-17A secretion in vitro.ResultsDAPT treatment alleviated the severity of IMQ-induced mouse psoriasis-like skin inflammation and decreased the scores of erythema, scaling and thickening. HE stain reveals obviously reduced epidermal hyperplasia and dermal inflammatory cells infiltration in IMQ + DAPT-treated mice. The increased expression of splenic Th17 cell percentage, along with Notch1, Hes-1, RORγt and IL-17A mRNA and IL-17A serum concentration in IMQ-treated mice were significantly decreased when experimental mice were treated by IMQ and DAPT combinedly. Data obtained from in vitro study in IMQ-treated mice also demonstrated that blocking Notch1 signaling by DAPT can result in a dose-dependent decrease of Th17 cell proportion, mRNA expression of Notch1, Hes-1, RORγt and IL-17A as well as IL-17A secretion in splenic CD4+ T cells.ConclusionThese data suggest that Notch1 inhibition by DAPT can effectively alleviate the severity of mouse psoriasis-like skin inflammation by regulating the differentiation and function of Th17 cells, indicating that DAPT might be a potential therapeutic candidate for the treatment of psoriatic inflammation.

TRAIL-Mediated Suppression of T Cell Receptor Signaling Inhibits T Cell Activation and Inflammation in Experimental Autoimmune Encephalomyelitis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cell apoptosis by transducing apoptosis signals after interacting with its receptor (TRAIL-R). Although the actual biological role of TRAIL remains to be elucidated, recent accumulating evidence implies that TRAIL regulates immune responses and immune cell homeostasis via an apoptosis-independent pathway, suggesting a novel immune-regulatory role of TRAIL in autoimmune diseases. The purpose of this study is to address the immune-regulatory role and molecular mechanism of TRAIL in regulating T cell activation in autoimmune diseases. TRAIL was administered to mice to induce experimental autoimmune encephalomyelitis (EAE), and to evaluate its impact on neuroinflammation and disease activity. The effects of TRAIL on neuroantigen [myelin oligodendrocyte glycoprotein (MOG)35-55]-activated T cell proliferation and cytokine production were investigated. TRAIL-treated MOG35-55-activated splenic Th17 cells were further adoptively transferred into Rag1 KO mice to induce passive EAE. Gene expression profiles of CD4+ T cells from EAE mice treated with TRAIL were analyzed by RNA sequencing and transcriptome analysis. TRAIL suppressed autoimmune encephalomyelitis and inhibited T cell reactivity to neuro-antigen in murine EAE, and the effects were dependent on TRAIL-R signaling. Moreover, TRAIL directly inhibited activation of MOG35-55-activated CD4+ T cells, resulting in suppression of neuroinflammation and reduced disease activity in adoptive transfer-induced EAE. Furthermore, TRAIL-R signaling inhibited phosphorylation of proximal T cell receptor (TCR)-associated tyrosine kinases in activated CD4+ T cells. Importantly, TRAIL/TRAIL-R interaction downregulated TCR downstream signaling genes in RNA sequencing and transcriptome analysis. TRAIL/TRAIL-R interaction regulates CD4+ T cell activation in autoimmune inflammation and directly suppresses T cell activation via inhibiting TCR signaling, suggesting that TRAIL-R serves as a novel immune checkpoint in T cell responses.

Modulation of Th17 and regulatory T-cell responses during murine pregnancy contributes to increased maternal susceptibility to Salmonella Typhimurium infection.

Salmonella Typhimurium (S. Tm) infection in pregnant mice results in massive placental infection, fetal loss, and exacerbated systemic infection. The Th17 host response can aid control of S. Tm infection, whereas successful pregnancy correlates to a dampened inflammatory and enhanced regulatory T-cell (Treg) response. Mice were infected systemically with S. Tm and tissue bacterial burden, splenic Th17 and Treg cell numbers, and serum cytokines were analyzed. Splenic and/or placental mRNA expression of IL-17A, RORγ-t, IL-10, and TNF was determined. The effects of in vivo CD25+ cell depletion and TLR4 blockade on the course of S. Tm infection and Th17 response were determined. Enhanced S. Tm burden in pregnant mice was associated with time-dependent increased serum inflammatory cytokines. In vivo, TLR4 blockade reduced splenic S. Tm burden, suggesting detrimental TLR4-mediated inflammation. However, the splenic and placental Th17 response was reduced in S. Tm-infected pregnant mice relative to non-pregnant controls. Alternatively, there was an increase in splenic Treg frequency in pregnant mice and depletion of this subset reduced bacterial burden and increased the Th17 response. Downregulation of Th17 cell responses by Tregs during pregnancy potentially contributes to exacerbation of S. Tm infection in pregnant mice.

Effect of Halofuginone on the Pathogenesis of Autoimmune Thyroid Disease in Different Mice Models

Our objectives were to investigate the therapeutic effect of halofuginone (HF) in the treatment of autoimmune thyroid diseases (AITDs) and explore its underlying mechanism of action. The Graves' disease (GD) model was generated by immunizing female BALB/c mice with adenovirus expressing the TSH receptor A subunit (Ad-TSHR289). The Ad-TSHRA+HF and Ad- TSHRA+DMSO groups were injected intraperitoneally with HF or the vehicle control (DMSO), respectively. The autoimmune thyroiditis (AIT) group consisted of female NOD.H-2h4 mice that were administered NaI in the drinking water and intraperitoneally injected daily with the vehicle control (DMSO) during the study period. The AIT/HF group consisted of female NOD.H-2h4 mice that were administered NaI in the drinking water and intraperitoneally injected daily with HF. The frequencies of splenic Th17 cells, Tregs and Bregs were determined by flow cytometry. The mRNA levels of IL-17, forkhead box P3 (Foxp3), RORγt and IL-10 were determined by real-time PCR. In both Ad-TSHRA+DMSO and Ad-TSHRA+HF groups, 10 out of 15 mice displayed serum T4 and TSAb levels above 3 SD beyond the mean control levels. The number of CD4+CD25+Foxp3+ T lymphocytes in the GD model was significantly increased in the HF group compared with the DMSO group (P < 0.05). The mRNA level of Foxp3 was significantly increased in the Ad-TSHRA+HF group compared with the Ad-TSHRA+DMSO group (P < 0.05). However, neither the abundance of CD4+IL-17+ T cell subpopulation nor the mRNA expression level of RORγt differed significantly between the Ad-TSHRA+HF and Ad-TSHRA+DMSO groups (P > 0.05). The serum TgAb titer was significantly reduced in the AIT/HF group compared with the AIT group (P < 0.01). The differences in the number of CD4+CD25+Foxp3+ T lymphocytes and the mRNA levels of Foxp3 between the AIT/HF and AITgroups were not significant (P > 0.05). However, the number of CD4+IL-17+ T cells and the mRNA levels of IL-17 and RORγt were significantly increased in HF-treated mice compared with the non-treated AIT-induced mice (P < 0.05). Treatment with HF significantly decreased the incidence of AIT by decreasing the number of CD4+IL-17+ T cells.

Modulation of Th17 and Regulatory T cell responses during murine pregnancy contributes to increased maternal susceptibility to Salmonella Typhimurium infection

PROBLEM Salmonella Typhimurium (S. Tm) infection in pregnant mice results in massive placental infection, fetal loss, and exacerbated systemic infection. The Th17 host response can aid control of S. Tm infection, whereas successful pregnancy correlates to a dampened inflammatory and enhanced regulatory T-cell (Treg) response. METHOD OF STUDY Mice were infected systemically with S. Tm and tissue bacterial burden, splenic Th17 and Treg cell numbers, and serum cytokines were analyzed. Splenic and/or placental mRNA expression of IL-17A, RORγ-t, IL-10, and TNF was determined. The effects of in vivo CD25+ cell depletion and TLR4 blockade on the course of S. Tm infection and Th17 response were determined. RESULTS Enhanced S. Tm burden in pregnant mice was associated with time-dependent increased serum inflammatory cytokines. In vivo, TLR4 blockade reduced splenic S. Tm burden, suggesting detrimental TLR4-mediated inflammation. However, the splenic and placental Th17 response was reduced in S. Tm-infected pregnant mice relative to non-pregnant controls. Alternatively, there was an increase in splenic Treg frequency in pregnant mice and depletion of this subset reduced bacterial burden and increased the Th17 response. CONCLUSION Downregulation of Th17 cell responses by Tregs during pregnancy potentially contributes to exacerbation of S. Tm infection in pregnant mice.